1. Field of the Invention
The present invention generally relates to an integrated, miniaturized 2-D IEF/PAGE analysis device provided in a microfluidic format. This invention further provides separation dimensions (IEF and PAGE) in a continuous gel format. More particularly, the invention provides a first dimension having an immobilized pH gradient for rapid and high resolution isoelectric focusing (IEF), and a second dimension having a plurality of microchannels for polyacrylamide gel electrophoresis (PAGE). The invention also provides a means for introducing an anionic surfactant such as sodium dodecyl sulfate (SDS) into the second dimension.
2. Background and Related Art
One of the most reliable separation techniques used in proteomics involves combining isoelectric focusing with polyacrylamide gel electrophoresis (IEF-PAGE). Samples are first separated based on their isoelectric points along an established pH gradient, then separated orthogonally by their molecular weight through a size-exclusion polyacrylamide gel. The result is a high resolution two-dimensional fractionation of all to species in the sample. The total peak capacity for a 2-D separation mechanism is the product of the peak capacities of each dimension, thereby significantly enhancing the assay resolving power: ntotal=n1n2. Conventional IEF-PAGE methodology involves using 7-18 cm-long immobilized pH gradient (IPG) strips for the first dimension, then transferring the strip to a slab gel for subsequent size-based elution. An anionic surfactant such as sodium dodecyl sulfate (SDS) is often included in the second dimension to enhance resolution by ensuring that all proteins obtain a constant charge/mass ratio. Unfortunately these assays are considerably labor-intensive and time-consuming, wherein the typical run time for a complete 2-D analysis can exceed 36 hours.
It will be appreciated that there has existed a long-felt need to miniaturize the 2-D IEF/PAGE process and incorporate it into a microfluidic format. Current techniques for deploying 2-D separation rely on handling bulk polymer gel slabs and/or performing a first separation process with one device and then moving the output of this first process to a second device for performing a second separation process (see U.S. Ser. Nos. 7,517,442, 6,969,452, and 6,013,165 and Published U.S. Application Serial Numbers 2002/0153252 and 2002/0033336). No one, however, has yet to provide a means for creating such gradients in microfluidic devices. Current methods suffer from several drawbacks, including complex instrumentation requirements, lack of temporal stability, and bulk flow along or perpendicular to the established gradient.
Implementation of IEF and PAGE methodology into a microfluidic format provides several advantages over conventional approaches, such as: 1) reduced run times due to shorter length scales, 2) reduced sample volume requirements, and 3) on-chip automation results in reduced user interaction. While modular microfluidic IEF and PAGE approaches have been extensively explored in the literature (see J. Wu and J. Pawliszyn, Electrophoresis, 1993, v.14(1): pp. 469-474; J. Han and A. K. Singh, Journal of Chromatography A, 2004, v1049(1-2): pp. 205-209; and W. Tan, Z. H. Fan, C. X. Qui, A. J. Ricco, and I. Gibbons, Electrophoresis 2002, v.23(20): pp. 3638-3645), synchronized “on-chip” integration of the two schemes remains elusive (see A. E. Herr, J. I. Molho, K. A. Drouvalakis, J. C. Mikkelsen, P. J. Utz, J. G. Santiago, and T. W. Kenny, Analytical Chemistry, 2003, v.75(5): pp. 1180-1187; C. A. Emrich, I. L. Medintz, W. K. Chu, and R. A. Mathies, Analytical Chemistry, 2007, v.79(19): pp. 7360-7366; C. Das, J. Zhang, N. D. Denslow, and Z. H. Fan, Lab on a Chip 2007, v. 7(12): pp. 1806-1812; and J. Liu, S. Yang, C. S. Lee, and D. L. DeVoe, Electrophoresis 2008, v.29(11): pp. 2241-2250).
One of the primary limitations for this integration is the effect of diffusion on band resolution within the free-solution IEF stage. While polyacrylamide IPG strips are used for conventional 2-D separations, immobilizing a pH gradient on a microfluidic device is a considerable challenge. Another significant challenge involves transferring the focused bands from the liquid phase to a gel for the secondary separation. Here, we disclose high-resolution, rapid, fully-automated microfluidic platforms for achieving 2-D separations. We recently demonstrated a technique for photopolymerizing precise and well-controlled microscale immobilized pH gradients on-chip (U.S. patent application Ser. No. 12/182,755, herein incorporated by reference: and G. J. Sommer, A. K. Singh, and A. V. Hatch., Analytical Chemistry, 2008, v.80(9): pp. 3327-3333). This publication represented the first successful implementation of IPG methodology onto a microchip, of which we are aware, which also provided resolving power comparable to that of macroscale IPG strips. Moreover, we have also recently provided a method for fabricating microscale isoelectric fractionation (pIEF) membranes as disclosed in commonly-owned, co-pending U.S. patent application Ser. No. 12/243,817, also herein incorporated by reference in its entirety. The device presented herein, therefore, can be fabricated to use either continuous μIPG gels, or the microscale isoelectric fractionation (pIEF) membranes we have also previously disclosed for the first dimension to yield an all-gel 2-D separation microdevice, and thus enabling simpler separations with higher resolution over similar devices reported in the literature.